Thin filament-associated actin-binding proteins control both actomyosin-based contractility and cytoskeletal formation in muscle and non-muscle cells. To elucidate these mechanisms, it is crucial to determine the structural interactions of the proteins involved. We address several interrelated questions fundamental to understanding muscle thin filament function: (1) What is the architecture of the thin filament in skeletal, cardiac and smooth muscles? (2) What are the changing structural interactions of thin filament-linked proteins that regulate muscle activity? (3) How do these proteins interact with actin to form the muscle cytoskeleton? We use state-of-the-art electron microscopy, image analysis and 3-D reconstruction to establish he macromolecular structure of components bound on thin filament actin. Using these techniques: (A) We aim to determine the structural basis of troponin-tropomyosin regulation of skeletal and cardiac muscle activity by analyzing interactions of tropomyosin and troponin on thin filaments and the effects of Ca2+ and myosin-crossbridge binding. (B) To help understand the organization of the cortical actin cytoskeleton of muscle cells, we aim to determine the structural interactions of dystrophin and utrophin with filamentous F-actin by examining the binding of their distinct calponin homology (CH)-domains. (C) We aim to assess the structural role of thin filament associated caldesmon as a possible modulator of actomyosin-based motility and cytoskeletal assembly in smooth muscle. (D) Studies of the structure of nebulin bound to actin will be part of our continuing investigation of the functional design of thin filaments. In each study, reconstructions will be fitted to the atomic resolution maps of F-actin to define molecular contacts of binding proteins with actin. Further, such "hybrid crystallography" will be used to fit newly solved crystal structures of troponin, tropomyosin, dystrophin and utrophin domains within EM density maps to attain near atomic resolution. Our ongoing studies on troponin-trepomyosin regulated filaments will lead to an elucidation of the molecular mechanism of relaxation and activation in skeletal and cardiac muscle. Our successfully initiated structural studies on utrophin and dystrophin will establish their unique features as cytoskeletal elements, information applicable to designing genetic therapies for muscular dystrophy. Studies on smooth muscle filaments will contribute to understanding the fine-tuning of the smooth muscle contractile response. Such modulation affects vascular tone and pulmonary airway resistance, determinants in, e.g., hypertension and asthma. The wider significance of our goals is underscored by the role of actin and associated proteins in diverse and vital cellular mechanisms that can become aberrant in malignancy.